Binding stability of peptides to Co-Cr-Mo alloy affects proliferation and differentiation of osteoblast.

Cobalt-chromium-molybdenum (CCM) alloys possess high corrosion-resistant properties as well as good mechanical properties. Hence, the alloys are employed in medical implants such as artificial knee and hip joints, coronary stents, and removable partial dentures. To improve the biocompatibility of CCM alloys, we reported that CCM-binding peptide (CBP) linked to cell-adhesive motif Arg-Gly-Asp (RGD) improved the attachment of endothelial cells on CCM alloys. However, the stability of CBP adsorption on the alloy and its effect on osteoblast compatibility are still unclear. In this study, we evaluated the stabilization of the adsorption layer of CBP-RGD on CCM alloy surface and investigated the effect of CBP-RGD peptide on the proliferation and differentiation of the osteoblasts. CBP-RGD layer exhibited higher stabilization than the RGD adsorption layer for 7 days. In addition, the proliferation of osteoblast on CBP-RGD adsorbed alloy higher than that on RGD adsorbed alloy. Moreover, the calcification of cells cultured on the CBP-RGD adsorbed alloy was significantly higher than that of the cells on RGD adsorbed alloy. These findings indicate that the CBP binding was stable during the culture of osteoblasts on the CCM alloy.

Post-synapse model cell for synaptic glutamate receptor (GluR)-based biosensing: strategy and engineering to maximize ligand-gated ion-flux achieving high signal-to-noise ratio.

Cell-based biosensing is a "smart" way to obtain efficacy-information on the effect of applied chemical on cellular biological cascade. We have proposed an engineered post-synapse model cell-based biosensors to investigate the effects of chemicals on ionotropic glutamate receptor (GluR), which is a focus of attention as a molecular target for clinical neural drug discovery. The engineered model cell has several advantages over native cells, including improved ease of handling and better reproducibility in the application of cell-based biosensors. However, in general, cell-based biosensors often have low signal-to-noise (S/N) ratios due to the low level of cellular responses. In order to obtain a higher S/N ratio in model cells, we have attempted to design a tactic model cell with elevated cellular response. We have revealed that the increase GluR expression level is not directly connected to the amplification of cellular responses because the saturation of surface expression of GluR, leading to a limit on the total ion influx. Furthermore, coexpression of GluR with a voltage-gated potassium channel increased Ca(2+) ion influx beyond levels obtained with saturating amounts of GluR alone. The construction of model cells based on strategy of amplifying ion flux per individual receptors can be used to perform smart cell-based biosensing with an improved S/N ratio.

Analysis of disordered abrasive scratches on titanium surfaces and their impact on nuclear translocation of yes-associated protein.

The morphology of the metallic surface of an implant is important for its contact with bone tissue as it directly affects osteoblast functions, such as cell adhesion, proliferation, and differentiation. Firm contact between the implant and cells creates a barrier that prevents inflammation and bacterial infections. Therefore, optimizing surface morphology, such as surface roughness adjustments, is essential to improving the adhesion between the implant and cells for successful tissue regeneration. However, the manner in which the cells sense the surface roughness and morphology remains unclear. Previously, we analyzed cell adhesion behavior and observed that inhibited cell spreading can delay osteoblast functions. Therefore, assuming that the surface morphology can be sensed through cell spreading, we investigated the cell spreading area and yes-associated protein (YAP) localization in mouse osteoblasts (MC3T3-E1) on a titanium surface with disordered abrasive scratches. Surface roughness of 100-150 nm was obtained by polishing, which inhibited the cell spreading, indicating that YAP localization in the nucleus was lower than that on other surfaces. The obtained results indicate that the cells sense the surface environment based on their spreading area, which regulates cellular functions via the Hippo pathway.

Transfection efficiency for size-separated cells synchronized in cell cycle by microfluidic device.

Non-viral system generally demonstrates less efficacious in transgene delivery than viral system; however it represents a safer alternative to viral system. In this study, transfection efficiency for human hepatocellular liver carcinoma cells synchronized in cell cycle at G0/G1 phase, which was sorted in size with a microfluidic device based on hydrodynamic filtration, was investigated by using a reverse transfection method. The synchronized cells were recovered at the yield of 80% from the micro-channel, and green fluorescent protein gene encoding plasmid mixed with lipofectoamine was transfected. The transfection efficiency of the cells at G0/G1 phase was 1.8 times higher than non-synchronized cells. The manipulation of cell cycle status could increase transfection efficiency in non-viral system, indicating size-based cell cycle synchronization is a powerful tool as a noninvasive method for bioscience and biotechnology.

Development of sensor cells using NF-κB pathway activation for detection of nanoparticle-induced inflammation.

The increasing use of nanomaterials in consumer and industrial products has aroused concerns regarding their fate in biological systems. An effective detection method to evaluate the safety of bio-nanomaterials is therefore very important. Titanium dioxide (TiO(2)), which is manufactured worldwide in large quantities for use in a wide range of applications, including pigment and cosmetic manufacturing, was once thought to be an inert material, but recently, more and more studies have indicated that TiO(2) nanoparticles (TiO(2) NPs) can cause inflammation and be harmful to humans by causing lung and brain problems. In order to evaluate the safety of TiO(2) NPs for the environment and for humans, sensor cells for inflammation detection were developed, and these were transfected with the Toll-like receptor 4 (TLR4) gene and Nuclear Factor Kappa B (NF-κB) reporter gene. NF-κB as a primary cause of inflammation has received a lot of attention, and it can be activated by a wide variety of external stimuli. Our data show that TiO(2) NPs-induced inflammation can be detected by our sensor cells through NF-κB pathway activation. This may lead to our sensor cells being used for bio-nanomaterial safety evaluation.

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array.

Engineered synapse model cell: genetic construction and chemical evaluation for reproducible high-throughput analysis.

Bioassay models of neural functions must lend themselves to high-throughput analysis in neural drug discovery. However, smart analysis methods for these functions have not yet been fully established. Here, we describe the development of a synapse model for cell-based biosensing. The engineered synapse model cell expresses ionotropic glutamate receptor on its surface, like the neural postsynaptic membrane. The advantages of the model cell are the ease of handling and reproducibility as compared with the cultured neural cell, and it can be employed to evaluate receptor function through ion flux analysis. The agonist-induced sodium influx was monitored as an agonist concentration-dependent increase in the observed fluorescence signal. Furthermore, we found that our model cell enables the correction of uneven cellular signal levels using a reporter system. Our engineered synapse model cell can be employed as a powerful tool for the screening of lead substances in pharmaceutical high-throughput analysis.

In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen.

Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ∼67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

Enzyme-based field-effect transistor for adenosine triphosphate (ATP) sensing.

Adenosine triphosphate (ATP) not only functions as an energy-carrier substance and an informative molecule, but also acts as a marker substance in studies of both bio-traces and cellular/tissular viability. Due to the importance of the ATP function for living organisms, in situ assays of ATP are in demand in various fields, e.g., hygiene. In the present study, we developed an ATP sensor that combines the selective catalytic activity of enzyme and the properties of an ion selective field effect transistor (ISFET). In this system, the ATP hydrolyrase, "apyrase (EC 3.6.1.5.)" is encased in a gel and mounted on a Ta(2)O(5) ISFET gate surface. When the enzyme layer selectively catalyzes the dephosphorylation of ATP, protons are accumulated at the gate because the enzymatic reaction produces H(+) as a byproduct. Based on the interfacial enzymatic reaction, the response from the ISFET is completely dependent upon the ATP concentration in the bulk solution. This device is readily applicable to practical in situ ATP measurement, e.g. hygienic usage.

Sub-micrometer scale surface roughness of titanium reduces fibroblasts function.

Titanium and its alloys are conventionally used to produce medical devices, but their biocompatibility has not yet been optimized. Surface modification, especially control of the surface roughness of titanium, is one strategy for improving biocompatibility and providing effective binding to hard tissue. However, the soft tissue compatibility of metallic materials is currently poorly understood, and effective techniques for tight binding between metal surfaces and soft tissue are still under development. Therefore, we here investigated whether the surface roughness of titanium affects fibroblast adhesion and proliferation. Our results showed that a surface roughness of ~100 nm reduces fibroblast function. On such surfaces, distinct focal adhesion was not observed. These findings improve the general understanding of the binding compatibility between soft tissues and metallic materials.

Quantum dots induce heat shock-related cytotoxicity at intracellular environment.

Quantum dots (QDs) are semiconductor nanocrystals with unique optical properties. Different proteins or polymers are commonly bound to their surfaces to improve biocompatibility. However, such surface modifications may not provide sufficient protection from cytotoxicity due to photodegradation and oxidative degradation. In this study, the cytotoxic effects of QDs, CdTe, and CdSe/ZnS were investigated using cadmium-resistant cells. CdTe QDs significantly reduced cell viability, whereas, CdSe/ZnS treatment did not markedly decrease the cell number. CdTe QDs were cytotoxic in cadmium-resistant cells suggesting that internalized QDs degraded and cadmium ions contributed to the cytotoxic effects. CdTe QDs were consistently more cytotoxic than CdSe/ZnS QDs, but both QDs as well as cadmium ions activated heat shock protein 70B' promoter. QDs themselves are likely to contribute to HSP70B' promoter activation in cadmium-resistant cells, because CdSe/ZnS QDs do not release sufficient cadmium to activate this promoter.

Microstructures and mechanical properties of Co-29Cr-6Mo alloy fabricated by selective laser melting process for dental applications.

The selective laser melting (SLM) process was applied to a Co-29Cr-6Mo alloy, and its microstructure, mechanical properties, and metal elution were investigated to determine whether the fabrication process is suitable for dental applications. The microstructure was evaluated using scanning electron microscopy with energy-dispersed X-ray spectroscopy (SEM-EDS), X-ray diffractometry (XRD), and electron back-scattered diffraction pattern analysis. The mechanical properties were evaluated using a tensile test. Dense builds were obtained when the input energy of the laser scan was higher than 400 J mm⁻³, whereas porous builds were formed when the input energy was lower than 150 J mm⁻³. The microstructure obtained was unique with fine cellular dendrites in the elongated grains parallel to the building direction. The γ phase was dominant in the build and its preferential <001> orientation was confirmed along the building direction, which was clearly observed for the builds fabricated at lower input energy. Although the mechanical anisotropy was confirmed in the SLM builds due to the unique microstructure, the yield strength, UTS, and elongation were higher than those of the as-cast alloy and satisfied the type 5 criteria in ISO22764. Metal elution from the SLM build was smaller than that of the as-cast alloy, and thus, the SLM process for the Co-29Cr-6Mo alloy is a promising candidate for fabricating dental devices.